JPH0253799A - Novel peptide and antimicrobial agent - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) expressed by the formula ((1) and (2) and (3)and (4) are respectively directly bonded; Arg-NH2 indicates that carboxyl group arginine is amide). USE:A medical antimicrobial agent, preservative or coating preservative. PREPARATION:For example, hematocytes of Limulus polyphemus that is a product of North America are added to a buffer solution at pH8.0, pulverized and centrifuged to collect precipitates, which are then added into 20mM hydrochloric acid, repulverized and centrifuged to provide a supernatant. The resultant supernatant is subsequently freeze-dried, redissolved, then subjected to column chromatography and eluted with 0.1% trifluoroacetic acid with acetonitrile concentration changed to 22-30%. The obtained column is then eluted with 28-30% acetonitrile and the eluted fraction is freeze-dried to afford polyphemusin I expressed by the formula.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides novel peptides with antibacterial activity extracted and isolated from North American horseshoe crab (Limulus mus) and salts thereof, and peptides containing these. The invention relates to antibacterial agents.

[Prior Art] In recent years, peptides have attracted attention as useful physiologically active substances. With the development of protein engineering,
In addition to the conventional natural peptides, research and development of synthetic peptides is also actively progressing, and the development of useful peptides has achieved considerable results so far, mainly in the pharmaceutical field. There is.

The development of peptides having antibacterial activity has been actively pursued in the past, and many reports have been made so far, mainly on relatively low-molecular-weight oligopeptides. For example, phosphonotripeptides that have antibacterial effects against Durham-positive bacteria and Durham-negative bacteria (Japanese Patent Application Laid-Open No. 57-106
689), phosphonodipeptide derivatives (JP-A-58-1
3594), cyclic peptide derivatives (JP-A-58-21
3744), 10 useful as antibacterial and antiviral agents.
A novel peptide consisting of the amino acid sequence of
51247), polypeptides useful as antibacterial agents against yeasts (JP-A-60-130599), glycopeptide derivatives useful as antibacterial agents against Durham-positive bacteria (JP-A-60-172998), novel glycopeptide derivatives (JP-A-60-172998);
JP-A-61-251699, JP-A-63-44598
), oligopeptides useful against Durham-positive bacteria (Japanese Patent Application Laid-Open No. 62-22798), peptide antibiotics (Japanese Patent Application Laid-open Nos. 6251697, 17897/1983), etc. have been reported.

[Problem that the invention is trying to solve]

However, there are quite a few of the conventional technologies that have not necessarily been put into practical use and are still beyond the realm of basic research, and it is understood that the development of peptide-based antibacterial agents is still in its infancy. In any case, the development of novel peptides that have excellent antibacterial activity and can be put to practical use as antibacterial agents is considered to be extremely important in establishing new application areas for peptides.

[Means for Solving the Problems] From this standpoint, the present inventors searched for various useful new peptides, and as a result, discovered that the North American horseshoe crab (L i
Extracted from the blood cells of mulus
The present invention was completed by discovering that the isolated peptide has a strong antibacterial effect.

That is, the present invention is recognized as including the technical matters described in (1) to (2) below.

1) It is represented by the following structural formula (1) (In the formula, ■ and ■, and ■ and ■ are each directly bonded. Arg NlI2 indicates that the carboxyl group of arginine is an amide.) peptide,
and its salt.

2) The following structural formula (I) (in the formula, ■ and ■, and ■ and ■ are each directly bonded. 8rg-NII2 indicates that the carboxyl group of arginine is an amide.) An antibacterial agent containing the expressed peptide and its salt as an active ingredient.

Thus, an object of the present invention is to provide novel peptides and salts thereof having strong antibacterial effects, as well as antibacterial agents containing these as active ingredients.

In this specification, this new peptide is referred to as Polyphemusin I. In addition, in this specification, IU per amino acid
PACIUB commission on Biol
They may be expressed using abbreviations based on logical nomenclature, and examples of these are as follows.

Arg: arginine, Trp: ) liptophan, Cy
s: 1/2 cystine, Phe: phenylalanine, Va
l: valine, Tyr: tyrosine, Gly nigericin, L
ys: lysine The novel polyphemusin of the present invention is 18
between each pair of cysteine molecules present at the 4th and 17th positions, and the 8th and 13th positions.
It has a bicyclic structure bridged by disulfide bonds. Further, the carboxyl group of arginine present at the C-terminus is amidated (-CONII2) and exists as arginine amide. Polyfuemcin I of the present invention
can be in the form of salts, for example salts with inorganic bases, organic bases such as alkali metal salts, alkaline earth metal ammonium salts, organic amine salts, and trifluoroacetic acid, methanesulfonic acid, hydrochloric acid. , addition salts of organic or inorganic acids such as sulfuric acid, nitric acid, and phosphoric acid.

The novel polyfemusin (1) and its salts of the present invention can be produced by the following method.

That is, a Tris-HCl buffer containing sodium chloride and benzamidine hydrochloride is added to the blood cells of a North American horseshoe crab (Limulus) to crush them, and the resulting mixture is centrifuged to obtain a precipitate. Add a hydrochloric acid solution to this, grind it, and centrifuge to obtain a supernatant. Add this to 5lhadex@G-50 and elute with hydrochloric acid solution. The elution fractions collected by measuring the absorbance at 280 nm were
By eluting with a trifluoroacetic acid solution added to No. 18 and varying the concentration of acetonitrile, both the desired polyfemsin I components are obtained.

Furthermore, polyfemusin I of the present invention can also be synthesized by a solid phase method. When this method is applied to the present invention, α−
The amino group of any amino acid is protected with a tert-butyloxycarbonyl group (Boci), and the hydroxyl group of tyrosine is protected with 2,6-dichlorobenzyljJ (C1z-
Bzl group), and the guanidino group of arginine is a tosyl group (
It is preferable to protect each with a Tos group). First, a protected derivative of the C-terminal amino acid arginine is introduced into a chloromethyl resin, and the amino acid chain is then sequentially extended to synthesize a protected peptide resin, which is then treated with hydrogen fluoride to cleave the protected peptide from the resin. , and simultaneously deprotected and reduced to obtain a synthetic peptide according to a conventional method. The obtained crude synthetic peptide was subjected to gel filtration, reverse phase HP
Purification is performed by means commonly used for peptide purification such as LC to obtain highly pure polyfuemcin I.

Regarding the solid-phase method commonly used for peptide synthesis, see, for example, Japan Biochemical Venue, "Biochemistry Experiment Course (■)" Protein Chemistry, Volume 4, pp. 208-495, (l@Tokyo Kagaku Doujin Publishing) It can be carried out according to the method of Merrifield et al. (1977) and Nobuo Izumiya et al., "Peptide Synthesis", published by Maruzen (1975).

The toxicity of Polyfuemcin I of the present invention is extremely low or non-toxic. When administering this polyfemucin I as a medical antibacterial agent, oral administration method or parenteral administration method,
That is, intravenous administration, intramuscular administration, subcutaneous administration, etc. are preferred. The amount of Polyfemucin (2) used per unit of administration is approximately 100 to 1000 (2). Furthermore, Polyfuemcin I of the present invention can be effectively used not only as a medical antibacterial agent but also as a preservative, an antibiotic, and a preservative for paints.

[Effects of the Invention] As shown in the Reference Examples below, Polyfuemcin I of the present invention exhibits a strong antibacterial effect against, for example, Gram-positive bacteria, Durum-negative bacteria, and fungi, and is useful as an inhibitor of these bacteria. . Specifically, it can be used in a wide range of fields such as medical antibacterial agents, preservatives, antibiotics, and paint preservatives, and these are all included in the scope of the antibacterial agents of the present invention. Needless to say.

(Examples) Hereinafter, examples of the present invention will be described to further explain the present invention in detail.

Example 1 (1) Production of Polyphemusin I Blood of North American horseshoe crab (Limulus)
37g of 20IIIM Tris-HCl buffer containing 50mM sodium chloride and 5mM benzamidine hydrochloride (
pl! 8.0) Add 100ml, crush, and centrifuge at 4°C (7500 rpm.

40 minutes) to obtain a precipitate. This operation was repeated twice to obtain a precipitate. Add 10% of 20mM hydrochloric acid solution to the obtained precipitate.
Add 0d and grind. Centrifuge at 4°C (7500 rpm)
40 minutes) to obtain a supernatant. Add 100 d of 20 rtM hydrochloric acid solution to the precipitate, grind again, and centrifuge at 4°C (7500 r
pm 40 minutes) to obtain a supernatant. The same operation was repeated two more times to obtain a supernatant. The resulting supernatant was collected and freeze-dried.

Dissolve the above lyophilized product in 20mM hydrochloric acid solution and hold at 4°C.
The supernatant was obtained by centrifugation (12,00 rpm, 15 minutes).

This was added to a 5ephadex @ G-50 (manufactured by Pharmacia) column (3 x 85 cm) and eluted with a 20 mM hydrochloric acid solution. While fractionating this into 10 ml portions, the absorbance at 280 nm was measured at the same time, and fraction number 66-
Both volumes of 87 were collected and freeze-dried. (See Figure 1).

This freeze-dried product was dissolved in 10 ml of 20 mM hydrochloric acid solution, and Cosmosil 05C+e (manufactured by Gyudon Chemical Co., Ltd.) column (1
0x 250 mm) and eluted with 0,1x trifluoroacetic acid solutions with varying concentrations of acetonitrile from 22 to 302. This elution curve is shown in FIG. This color 1, 28 to 30 x acetonitrile °? Polyfimucin I of the present invention was obtained by collecting and freeze-drying the elution fraction. Yield was 20 mg.

The infrared absorption spectrum of this polyfemsin I is as shown in Figure 3, 3,300.1650.1540.14
There are absorptions at 20.1240 and 740 cm-'.

Moreover, the ultraviolet absorption spectrum is as shown in FIG. The amino acid composition of this polyfemucin I is shown in Table 1.

Table 1 (2) Structure determination The structure of this substance was determined as follows.

That is, a sample in which disulfide bonds were cleaved using dithiothreitol and pyridylethylated with 4-vinylpyridine was hydrolyzed using 6N hydrochloric acid to determine the amino acid composition. In addition, this sample was subjected to 17 Edman degradations using a gas phase sequencer to determine the type and bonding order of amino acids from the N-terminal arginine to the 17th cysteine. In addition, the elution time on reversed-phase high performance liquid chromatography of the phenylthiocarbamyl derivative obtained by treating the aminoethylated sample with lysyl endopeptidase to release the C-terminal residue was determined separately. It was consistent with that of phenylthiocarbamyl argininamide. Furthermore, since the mass spectrometry results also showed a mass number in which the C-terminus was arginine amide, it was determined that the C-terminus was arginine amide.

Example 2 Formulation of Polyfemucin I An example of a formulation for oral or parenteral administration containing the polyfemucin I active ingredient of the present invention is shown below.

(1) 200mg tablet polyfemsin I 200mg cornstarch 40+ng lactose
98mg Magnesium Stearate
8 mg talc 4■ (2)
100mg ampoule for injection (q, s, p ampoule) Polyfemcin I 100mg Injection water Appropriate amount (3) 500mg capsule Polyfemcin 1 500mg lactose
50 ■ Magnesium stearate 5 mg (4) 500 mg tablet Polyfemsin I 500 mg corn starch 10 mz polyvinylpyrrolidone
35mg lactose 7
4 mg Magnesium stearate 14 mg Talc 7 mg Below, the results of an investigation on the antibacterial activity of the peptide borifuemucin I of the present invention are shown as reference examples.

Reference example (+) Antibacterial test against Gram-positive bacteria and Durham-negative bacteria ■ Experimental materials Sta h 1ococcus aureus AT
CC25Bacillus 5ubtilis IA
M 1069 medium: Bovine heart exudate 250g Casamino acid 15g I,-tryptophan 0.05g Purified water 10100O ■Experimental method Harmful bacteria were cultured with shaking in Nutrient BrothO (manufactured by Dico) medium for 20 hours, and the concentration of the bacteria was determined. 660n
Adjust the absorbance of m to about 0.3, and then
A test bacterial solution was prepared by adding mρ to the above medium. Dissolve 300 μg of the lyophilized product of this substance in 12 m2 of purified water and
A solution of μg/d is obtained. This was used as a stock solution and was diluted 2 times stepwise to give 12.5.6.25.3.13.1.56 μg
/ml sample solution was obtained.

1 ml of each concentration of sample solution was dispensed into sterilized test tubes, 1 ml of each bacterial solution was added thereto, mixed, cultured at 37°C for 20 hours, and the absorbance at 660 nm was measured. As a control, purified water was used instead of the sample solution, and the minimum growth-inhibiting concentration of this substance for each fraction was determined. The results are shown in Table 2.

(Left space below the wooden page) Table 2 Salmonella b.
ccus anreus ATCC259233,13 6,25 (2) Antibacterial test against fungi ■Experimental materials Test bacteria: Yamahigakure Psnicillium Medium: Polypeptone malt extract Glucose agar Purified water 11 animals funiculosum 1g 0g 0g 0g 000ml ■Experiment Method: 50011 g of the lyophilized product of this substance was dissolved in 2 ml of purified water to obtain a sample solution with a concentration of 250 μg/d. This was diluted in two-fold steps to prepare a sample solution of 125.62.5.31.3 ug/ml.

1 micron of each concentration of sample solution was added to the above-mentioned culture medium 9, and 5 rrrl of this was dispensed into sterilized test tubes to prepare a slant culture medium. A straight line was drawn on this slant medium with a platinum loop to which conidia of the above bacteria were attached, and cultured at 30'C for 48 hours.
I investigated its growth. The results are shown in Table 2.

Figure 2 shows the elution curve of acid solution extract, Figure 2 shows the elution curve of 0 and 1% trifluoroacetic acid solution on Cosmosil ■5 CIll column, Figure 3 shows the elution curve of polyfemsin I.
Figure 4 shows the infrared absorption spectrum of polyfemsin I.
It is a figure showing the ultraviolet absorption spectrum of.

Table 3

Claims (1)

[Claims] 1) The following structural formula (I) [There is a gene sequence] (In the formula, [1] and [2], [3] and [4] are directly bonded to each other. Arg-NH_2 indicates that the carboxyl group of arginine is an amide.) A peptide and a salt thereof. 2) The following structural formula (I) [There is a gene sequence] (In the formula, [1] and [2], [3] and [4] are each directly bonded. Arg-NH_2 is arginine An antibacterial agent whose active ingredient is a peptide represented by (indicates that the carboxyl group of is an amide) or a salt thereof.